Long-Term Expression of the Biologically Active Growth Hormone in Genetically Modified Fibroblasts after Implantation into a Hypophysectomized Rat

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Abstract
We employed the hypophysectomized rats as an animal model to explore the feasibility of using genetically engineered fibroblast cells for growth hormone gene therapy. An internal ribosome entry site (IRES)-directed bicistronic retroviral vector, PSN, which contained a porcine growth hormone (pGH) cDNA at the first cistron and a Neor gene at the second cistron was used to infect primary rat embryo fibroblast (REF) cells. The infected cells (5 × 106 cells/rat) were injected directly into the peritoneum of syngeneic hypophysectomized rats. We demonstrate that the implanted PSN-infected REF cells could secrete biologically active pGH in vivo, leading to significant growth of the tibia at day 15 and day 57 post-implantation. We also treated the PSN-infected REF cells with collagen to form a tissue-like structure. The skin-like discs were grafted underneath the skin on the back of rats and cells were retrieved at different times. Using two criteria, semiquantitative reverse transcription-polymerase chain reaction on the pGH RNA extracted from the explants and G418 resistance conferred from the explanted cells, we demonstrate that pGH was expressed in the implanted fibroblasts up to 70 days. Despite the fact that the total pGH RNA level was reduced in the explants of longtime post-implantation, which was probably due to the reduction of transduced cells retained in the explants, the specific efficiencies of pGH RNA expression from these explants were maintained as high as the primary PSN-infected REF prior implantation. These results suggest that fibroblast cells are capable of expressing the foreign genes persistently in vivo. Primary fibroblast cells are the potential target cells for transducing secretory proteins for gene therapy purpose because of their easy access to the circulation. In this paper, we demonstrate a sustained expression of porcine growth hormone (pGH) from the implanted rat embryo fibroblast cells for at least 70 days in an animal model of growth hormone deficiency. These results, as improved from previous findings, might raise issues regarding careful evaluation of primary fibroblast cells for human gene therapy.